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Original Studies |
in Human and Rabbit Skeletal Tissues
Medical Research Council Bone Research Laboratory, University of Oxford, Nuffield Orthopedic Center, Oxford, United Kingdom OX3 7LD
Address all correspondence and requests for reprints to: Dr. James T. Triffitt, Medical Research Council Bone Research Laboratory, Nuffield Department of Orthopedic Surgery, University of Oxford, Nuffield Orthopedic Center, Oxford, United Kingdom OX3 7LD.
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Abstract |
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, to particular cells to test the hypotheses that 1) estrogen
directly influences growth plate chondrocytes; and 2) estrogen has a
principal action on bone tissue via osteoblasts. ER messenger
ribonucleic acid (mRNA) was localized by in situ
hybridization in human specimens from five males (11–15 yr old), two
females (9 and 11 yr old), and three growing rabbits. In all of the
human material examined, ER mRNA was consistently identified in
chondrocytes. In all of the rabbit tissue studied, ER mRNA was
localized in chondrocytes of the growth plate and the subarticular
epiphyseal growth center. ER mRNA signals were readily observed in
both active osteoblasts and lining cells on trabecular surfaces of all
samples. No clear evidence of positive staining was detectable in
osteoclasts or osteocytes in either species. The distribution of ER
mRNA coincided with immunolocalization of the ER protein in the human
specimens. These data suggest a direct action of estrogen on growth
plate chondrocytes that may affect longitudinal growth and subsequent
fusion of the growth plate and also on osteoblasts to affect bone
formation at trabecular sites. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Until recently, only one "classical" form of the ER was known to
exist. With the discovery of a new form of the receptor, ERß, (6) the
original form is now identified as ER. ERß has been shown to be
expressed in human thymus, spleen, ovary, and testis, and recently, its
expression in osteoblasts from rat bone (7) has been described. The
results of studies on the mechanism of action of estrogen on the
skeleton have been controversial. After initial evidence of
localization of ER in osteoblast-like cell lines (8, 9), there have
been only isolated reports of localization in untransformed mammalian
bone cells ex vivo using immunohistochemical techniques
(10, 11, 12). In light of the findings implicating ER activity in skeletal
growth, we have applied in situ hybridization procedures
with digoxigenin-labeled human ER riboprobes to specimens from young
human and rabbit bone tissues. Immunohistochemical procedures were
employed to confirm the distribution of ER protein in the human
specimens. We hypothesized that localization of ER to particular cell
types would suggest likely pathways of estrogen action in
vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue samples were obtained from seven human patients and three
New Zealand White rabbits, as detailed in Table 1
. Human bone was obtained during
epiphyseodesis for corrective treatment of leg growth by curettage of
the growth plate, and the fragmented specimens were fixed immediately
in 4% (wt/vol) paraformaldehyde in phosphate-buffered saline (PBS; pH
7.4). Part of the entire growth plate was obtained from patient 4 after
leg amputation for surgical treatment of malignant disease. This tissue
was obtained as a normal pathological specimen and had been fixed
routinely in formalin. Only human tissues that otherwise would have
been discarded were used, with the approval of the hospital medical
staff committee. Rabbit long bones were rapidly removed after killing,
cut into small pieces, and fixed in 4% (wt/vol) paraformaldehyde in
PBS.
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). Mineralized tissues after dehydration in acetone and
xylene were embedded in a resin mixture containing methyl methacrylate
and dibutylphthalate (4:1, vol/vol), polyethylene glycol 400 (1.2%,
vol/vol), N,N-dimethylaniline (0.6%, vol/vol),
and benzoyl peroxide (2%, wt/vol). Resin was polymerized at 4 C
overnight, and embedding was completed at room temperature. Before
paraffin embedding, decalcification in ethylenediamine tetraacetic acid
(15%, wt/vol) for 7 days was performed, followed by dehydration in
alcohol and xylene. Sections (4–5 µm) were placed on
3-aminopropyltriethoxysilane-coated slides, and adjacent sections were
processed for evaluation by in situ hybridization procedures
or routine histology. Preparation of riboprobes
The complementary DNA plasmid used for detecting ER was a
gift from P. Chambon (Strasbourg, France). The original plasmid
contained a 1.8-kb fragment of the human ER-coding region in pSG5
vector. A 1140-bp 3'-fragment was subcloned into pGEM 7Z+
for generating riboprobes. Before generating the riboprobes, plasmids
were linearized with the appropriate restriction endonucleases to give
sense and antisense strands. Probes were labeled with digoxigenin using
a kit (Boehringer Mannheim, Mannheim, Germany) according to the
manufacturer’s instructions. The specificity of the ER antisense
probe was assessed by Northern analysis using messenger ribonucleic
acid (mRNA) isolated from estrogen-responsive MCF-7 breast cancer
cells, as described previously (13).
In situ hybridization procedure
Resin was removed from the sections with acetone and paraffin
with Histoclear (National Diagnostics, Atlanta, GA). Deproteinization
was carried out by the use of hydrochloric acid (0.2 mol/L), followed
by digestion with proteinase K. Subsequently, sections were refixed
with paraformaldehyde (4%, wt/vol) and acetylated using acetic
anhydride (0.25%, vol/vol) in triethanolamine (0.1 mol/L; pH 8.0).
Slides were rinsed in PBS between each treatment step. Antisense or
sense probe was applied to each of two sections on each slide to ensure
identical subsequent treatment conditions. Hybridization was performed
overnight at 60 C for ER mRNA detection. Posthybridization treatment
included digestion of unbound probe with ribonuclease and washes with
SSC (1, 0.5, and 0.1 x). Detection of hybridized probe was carried out
with alkaline phosphatase-coupled antidigoxigenin antibody according to
the manufacturer’s instructions (Boehringer Mannheim). Human breast
tissue was used as positive control for ER mRNA, and tissue
preservation of RNA was assessed by in situ hybridization of
oligo(deoxythymidine) [oligo(dT)] probe (R&D Systems, Abingdon, UK)
in representative sections from all specimens. All photographs were
taken under differential interference contrast microscopy [Axiophot,
Carl Zeiss (Oberkochen), Garden City, Herts, UK] to emphasize cellular
morphology. No differences were observed in the results from in
situ hybridization using the plastic- and paraffin-embedded
tissues.
Immunohistochemistry for ER
Localization of ER was also analyzed by immunohistochemistry on demineralized paraffin-embedded specimens. Paraffin-embedded breast tissue and cryosections of human bone were used in control procedures. A standard indirect peroxidase procedure recommended by the manufacturer was followed using concentrated monoclonal mouse antibodies to human ER and reagents from Biogenex (San Ramon, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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riboprobe
Figure 1 indicates the specificity
of the ER antisense riboprobe with a single major band at
approximately 6.6 kb on a Northern blot analysis of mRNA from MCF-7
cells.
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Tissue samples obtained from all patients except one (Table 1)
contained pieces of bone and growth plate cartilage. The sample from
patient 4 included a portion of the entire epiphysis and some
metaphysis. In each of the human specimens, several cartilage pieces of
different sizes were observed, all containing ER-positive cartilage
cells. As assessed by morphological appearance, they originated from
the resting, proliferative and hypertrophic zones of the growth plate
(Fig. 2, a and b). Material from patient
4, which contained a longitudinal section of the growth plate,
demonstrated the localization of ER mRNA in all zones of the growth
plate (Fig. 2d). Pairs of tissue sections hybridized with the sense
probe for ER mRNA showed no staining (Fig. 2c).
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, a and b) and osteoblasts
(Fig. 3c) and were positive for ER mRNA. Almost all of the bone
trabecular surfaces of the specimen of patient 4 contained osteoblasts
expressing ER mRNA (Fig. 2d). Osteocytes (Fig. 3b) and osteoclasts
(Fig. 3d) did not express detectable levels of ER mRNA. Similar
findings concerning the localization of ER were found in all human
specimens, and no differences were observed between those from male and
female patients.
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, a
and b).
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Localization of ER protein in human specimens by
immunohistochemistry coincided with that for ER mRNA expression. ER
protein was detected in osteoblasts (Fig. 2, e and f), osteoblast
progenitors (Fig. 2e), bone lining cells, and growth plate
chondrocytes, but not in mature osteocytes or osteoclasts (Fig. 2f). No
differences in localization were found in frozen sections or those
obtained by demineralization and paraffin embedding (not shown).
Rabbit tissues
The long bone specimens from the three rabbits (Table 1) were
found to contain cells positive for ER mRNA. Compared to that in the
rabbit specimens, the intensity of the signal in human osteoblasts was
stronger. Each rabbit long bone specimen examined consisted of the
growth plate (physis) and articular cartilage, including the epiphyseal
and metaphyseal bone tissue. Positive staining was observed in
chondrocytes of the growth plate and subarticular epiphyseal growth
center (Fig. 5). In the growth plate,
hypertrophic chondrocytes mostly two to four cells high, in some places
more, next to trabecular bone showed strongly positive signals for
ER mRNA (Fig. 5, a and b). This band of positive cells was located
across the lower hypertrophic zone. Paired sections treated with the
sense probe showed no staining (Fig. 5c).
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-positive cells
appeared in groups along the whole length of the articular cartilage
layer, but did not spread throughout the cartilage thickness (Fig. 5, d
and e). No positive staining could be seen in tissue sections
hybridized with the sense probe (Fig. 5f). All of the rabbit bone
specimens analyzed for expression of ER mRNA showed a similar
pattern of positive cells in the areas of the growth plate and
subarticular epiphyseal growth center.
Hybridization signals of lesser intensity were observed on most
osteoblast-covered, trabecular bone surfaces. The staining was
predominantly evenly distributed over cuboidal osteoblasts, in some
instances revealing a clear nucleus with a dark ring of cytoplasm (Fig. 6a). This could also be seen on the
primary trabecular bone surfaces in the proximity of the growth plate
(Fig. 6b). There was no staining of osteoblasts in the sections treated
with sense probe (Fig. 6, d and f). Neither mature osteocytes (Fig. 6c)
nor osteoclasts (Fig. 6e) expressed detectable levels of ER
mRNA.
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, c and d). However, in
the proliferative and maturation zones of the growth plate, no mRNA was
found in any specimens from the three rabbits examined (Fig. 4c). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in bone and
cartilage cells derived from both human and rabbit skeletal systems.
ER mRNA was localized in chondrocytes of the growth plate from all
seven human patients and the three rabbits studied. Additionally, ER
gene expression was detected in the subarticular epiphyseal growth
center of the rabbit long bones. These findings support and extend the
results of previous investigations that have examined estrogen effects
on the growth plate. Previous studies of ER protein localization using
immunocytochemical techniques have shown its presence within
proliferative and hypertrophic chondrocytes from human cartilage
derived from fetuses of 10–22 weeks gestation (14).
A recent preliminary communication reported ER localization above the
growth plate of an 18-month-old pig (12). The data presented in this
report demonstrate the presence of ER mRNA in the growth plate of
growing humans and rabbits. In particular, ER mRNA has been
demonstrated in the cartilage of all five boys studied, in all zones of
the growth plate. In rabbits, oligo(dT) hybridization studies of the
growth plate samples revealed preservation of mRNA in the hypertrophic
zone of the growth plate, but not in the proliferative zone. This may
explain our negative results for the presence of ER in the
proliferative zone, which may have resulted from vigorous ribonuclease
activity in this region and poor penetration of the fixative. The
immediate fixation of fragments of human tissue in ice-cold
paraformaldehyde may have overcome this problem.
The biological importance of ER in the growth plate is underlined by in vitro studies showing that estrogen promotes growth of the rabbit cartilage at specific stages of development of the growth plate (15). Recently, the effects of interruption of estrogen action by a disruptive mutation in the classical ER in a man aged 28 yr have been reported (4). A remarkable feature in this case is that bone maturation age is delayed, and the knee growth plates remain open. It has been recognized for many years that estrogen plays an important role in the growth and eventual fusion of the growth plate in girls (16, 17). It now appears that estrogen plays an important role in the fusion of the growth plate in boys. Significantly, our findings support the hypothesis that the effects of estrogen in males could involve direct, rather than indirect, actions on the growth plate chondrocytes.
In this study the presence of ER mRNA has been demonstrated in the
osteoblasts and lining cells of the trabeculae of male and female human
and rabbit bone in untransformed mammalian tissue ex vivo.
By immunohistochemistry, ER protein was also detected in osteoblasts
and lining cells in cryosections and in paraffin-embedded human bone
sections.
A previous report, using an immunocytochemical technique, found receptor protein localization in osteoblasts from calvarial bone from children, but no histological data were presented (10). There are several reports of ER localization using a variety of techniques in normal and transformed osteoblast-like cells (8, 9, 18, 19) and avian osteogenic cells (20, 21, 22). This report extends the data on possible estrogen effects on osteoblasts to cells present on the trabecular surfaces of bone in the growing skeleton. The importance of the effects of estrogen on growing bone in males is indicated by the reduction in bone mass in addition to absence of growth plate fusion in the male with a disruptive mutation in the ER (4). This suggests an important role for estrogen in achieving normal peak bone mass.
The negative findings in relation to the detection of ER mRNA in
both human and rabbit osteoclasts is interesting in light of the
positive findings in osteoblasts and chondrocytes in the same material
and the preservation of mRNA in all of these cells, as shown using the
oligo(dT) probe. This distribution was confirmed in the human specimens
by immunohistochemical localization studies. However, it is clearly
possible that the numbers of ER mRNA ribosome copies per cell and
the receptor numbers in the mature osteoclasts and osteocytes are below
the detection limits of both methods. The issue of sensitivity has been
raised in a recent publication (23), in which no detectable levels of
the ER mRNA in osteoclasts and osteocytes with low level signal in
osteoblasts was reported using conventional in situ
hybridization in the hands of these investigators. From our studies,
this is clearly not a problem, as significant signals are observed in
osteoblasts and chondrocytes by in situ hybridization using
riboprobes. The presence of the ER mRNA in osteoclasts and osteocytes
was identified by Hoyland et al. (23) by amplifying the
extremely low copy number of the target mRNA using in situ
RT-PCR to the point of detection. These data, however, are not in
agreement with the earlier findings that ER protein is immunolocalized
in only some osteocytes (12). This latter observation is more in
agreement with our finding that ER mRNA and protein are absent from
mature osteocytes. The very low copy number of the ER mRNA in mature
osteoclasts raises questions of the relative importance of a direct
action of estrogen through stimulation of this receptor in the
osteoclast compared to that in the osteoblast. Previous reports have
identified ER in osteoclast-like cells from digests of calvarial
bone from children (11), avian osteoclasts (24), and a preosteoclastic
cell line (25). ER mRNA has been found in osteoclast populations
from human giant cell tumor of bone in one report using Northern
analysis (26), but not in another using in situ
hybridization (27). In a study of eight human giant cell tumors, ER
protein was detected by Western blotting, and estrogen-binding sites
were shown in seven of eight specimens, but none was detectable by
immunohistochemistry (28). However, the relevance of the latter data to
the physiology of normal bone is uncertain, as 17ß-estradiol has been
shown to stimulate the bone resorptive activity of isolated rat
osteoclasts only in the presence of osteoblasts (29). Furthermore,
recent studies on the effects of estrogen or antiestrogens on avian
osteoclasts do not support the hypothesis of a direct effect on
osteoclasts by these compounds (30). It is noteworthy that induction of
the differentiated osteoclastic phenotype by phorbol ester treatment of
a preosteoclastic cell line leads to the loss of ER expression
(25).
Recent studies on the newly described isoform of ER, ERß, offer other
possible explanations for the direct effects of estrogen on target
tissues that do not exhibit the presence of ER. For example, ERß
has been located in ovaries and prostate, tissues that are not known to
contain ER (31, 32). Similarly, osteoclasts and mature osteocytes,
which showed a relative absence of ER in the present study, may also
express ERß and thus be directly responsive to estrogen via receptor
mechanisms. ERß has now been reported to be expressed in the human
osteoblastic cell line SV-HFO (33), rat calvarial primary osteoblasts,
and the rat osteosarcoma cell line ROS 17/2.8 (7). mRNA expression for
ERß was higher in rat and human osteoblasts in cell cultures than
that of ER mRNA. These findings imply that ERß receptors may be
involved in modulating estrogen action at an even more sensitive level
than ER in these cells. However, in a study on the transcriptional
activities at a classical estrogen-responding element and an activator
protein-1 element, ER and ERß receptors exhibited opposite signals
(34); 17ß-estradiol activated transcription with ER, but inhibited
transcription when the ligand was ERß. Other known antiestrogens
(tamoxifen and raloxifen) were demonstrated to activate transcription
with ERß at an activator protein-1 site. This new knowledge reveals a
high degree of complexity involved in estrogen action at the gene
transcription level.
In conclusion, the localization of ER mRNA in the growing tissue of
the growth plate and in osteoblasts suggests that these cells are the
targets for estrogen action in skeletal tissue during postnatal growth
and development. These findings may point to a continuing osteogenic
effect of estrogen in the mature skeleton in addition to its role in
the prevention of osteoclastic bone resorption. Further studies are
required to determine the relative expression of ER and ERß in
skeletal tissues in situ and the interactions and mechanisms
of action of estrogen signaling in skeletal cells.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Medicine, University Western
Australia and Sir Charles Gairdner Hospital, Perth, Western
Australia, Australia.
Received January 13, 1998.
Revised April 1, 1998.
Accepted April 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß mRNA during
differentiation of human osteoblast SV-HFO cells. Endocrinology. 138:5067–5070. and ER ß
at AP1 sites. Science. 277:1508–1510.This article has been cited by other articles:
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